The primary research focus of this grant is the elucidation of the role(s) of clathrin coated vesicles (CVs) in the exocytic transfer of newly synthesized calsequestrin (CSQ) and acetylcholinesterase (AChE) to the sarcoplasmic reticulum (SR) of developing skeletal muscle and in the transfer of newly synthesized proteins destined for rapid axonal transport to the nerve endings of the central nervous system. We have recently demonstrated that CSQ and AChE, both N-linked glycoproteins, are found in the CVs and SR in a form succeptible to cleavage by Endoglycosidase H and precipitation by Wheat Germ Agglutinin. The kinetics of their passage through CVs and the above data are consistent with a CV mediated transfer between the early intermediate Golgi apparatus and the SR. During the next grant period, we will precisely identify the oligosaccharide found on CSQ and search for a putative receptor for CSQ in the terminal SR cisternae and in CVs. We will also localize CSQ and two other major proteins of the SR, the calcium ATPase and the high mannose glycoprotein, using immunocytochemical techniques. With respect to the rapid transport process, we have recently isolated a highly enriched population of rapid transport vesicles from rabbit optic nerve after 35S-methionine injection into the rabbit eye. We will further characterize these vesicles and identify specific proteins e.g. substance P, glucose transporters, AChE, kinesin, which will allow identification of rapid transport vesicles from bovine optic nerve and cholinergic axon tracks. Once we have isolated rapid transport vesicles in large amounts we will characterize their interactions with kinesin and microtubules. We will also immunoisolate labelled coated vesicles subtypes from the retina to, hopefully, confirm the hypothesis that coated vesicles are precursors to rapid transport vesicles. We will also further characterize a CSQ like protein we have recently identified in brain, by biochemical and immunocytochemical methods. Our long term goal is to apply the knowledge we gain on the control of cytosolic Ca++ and transport of cholinergic enzymes to increase our understanding of the underlying cause of Alzheimer's disease. There are several recent reports suggesting a Ca++ deficit in cells from patients with Alzheimer's disease.